Recent development of fluorescent indicator dyes for biologically important intracellular components has made it possible to follow the time-dependent distribution of these components in intact cells. For example, see U.S. Pat. Nos. 5,049,673, 4,849,362 and 4,603,208.
The emitted fluorescent radiation from a fluorophore in response to excitation of a single wavelength generally includes a broad band of wavelengths. Spectra of emissions from different fluorophores will generally overlap. Also, when using radiations of different wavelengths to excite each of two fluorophores, their emission spectra may still overlap.
Fluorophores absorb excitation radiation at more than one wavelength. The absorption spectra of different fluorophores may overlap. As a consequence, radiation of a wavelength chosen to efficiently excite a certain fluorophore will also to some extent excite other fluorophores. There may occur spectral overlappings between these cross-excited emission spectra of other fluorophores and the emission spectrum of the fluorophore predominantly excited by the chosen wavelength. In summary, in many applications involving multiple fluorophores the fact that their emission spectra generally overlap and that their absorption spectra also may overlap constitutes a major problem. Making simultaneous quantitative measurements of the individual fluorophores is difficult or impossible. Therefore, in the past, simultaneous fluorescent detection has been primarily limited to cases where the wavelength regions of spectral overlap of the emissions can be suppressed by optical filtering. Then separation is achieved at the expense of losing valuable signal intensity.
The different emission spectra of a single fluorophore obtained by exciting it with different wavelengths generally overlap heavily and can not be separated by optical filtering. The relative intensities of these spectra depend on the shape of the absorption spectrum. This shape may convey valuable information about the fluorophore and its environment. Thus, there is a need to separately measure the cross-excited emission of a fluorophore and the main emission, thereby collecting information about the shape of the absorption spectrum. To achieve this in the case when two different fluorophores are present and two excitation wavelengths are simultaneously applied, the individual contributions from each fluorophore must be measured separately. This can not be done by optical filtering exclusively.
When a fluorophore is excited by light having a sinusoidally modulated intensity, the fluorescence emitted is also sinusoidally modulated. The modulation frequencies are the same but the phase of the emitted fluorescence is shifted by an amount related to the lifetime of the fluorophore's excited state. Mitchell U.S. Pat. No. 4,937,457 discloses a frequency domain spectrofluorometer which uses a single wavelength of excitation modulated at multiple harmonically related phase-locked frequencies to simultaneously determine the spectral response and phase shift of a single fluorophore to the entire range of modulation frequencies employed. The data produced is used to determine the fluorescence lifetime of a fluorophore.
In Takahashi U.S. Pat. No. 5,032,714 et al., a light waveform measuring device is used for measuring the lifetime of fluorescent light produced due to pulsed laser excitation. Two laser beams of different frequencies, at least one of which is pulsed, are used to produce a single-frequency pulsed beam selected from the sum frequency mixing of the beams. The output beam is pulsed at the same rate as the pulsed input beam which is used to trigger a single-photon detector or streak camera. The detector is thereby synchronized to the exciting beam.
Identification and discrimination of multiple fluorophores in a sample is disclosed in Loken U.S. Pat. No. 5,047,321 et al. Each component must have a distinguishable characteristic peak emission wavelength at which a detector is set. Fluorophores may be excited with a single wavelength or multiple wavelengths, but detection occurs in regions where the peak emission spectra do not overlap.
In Gardell U.S. Pat. No. 4,628,026 et al., an automated system for the sequential and alternate irradiation of a specimen by two distinguishable wavelengths of light is disclosed. The system classifies specimens based on the quotient of the fluorescent light intensities sequentially received from the specimen in response to the two excitation wavelengths.
Simultaneous recording of multiple fluorophores excited by a single wavelength using spectral filtering or separation is disclosed in Robertson, Jr. U.S. Pat. No. 4,833,332 to et al. The fluorophores which have overlapping emission spectra are distinguished by the ratio of their emissions transmitted by two spectral filters having complementary transmission spectra. The system is not capable of quantitative determinations.
The prior art devices which measure fluorescence at only one peak emission wavelength are unable to simultaneously quantify multiple fluorophores using the total emission from each fluorophore. Those devices which rely on spectral separation to distinguish multiple fluorophores are unable to separate the total contribution of each fluorophore from the combined emission spectrum detected.
Use of lock-in amplifiers for signal detection is known. Using a two-phase, rather than a single-phase lock-in amplifier, the value of the phase angle can be derived from the outputs of the in-phase and the quadrature channels of the amplifier. This has been used, for example, in cytometry by Steinkamp and Crissman (1992) to distinguish between fluorophores that have different decay times. It has also been proposed by Morgan et al. (1992) that this technique should be used in a confocal scanning microscope to produce images that represent the decay time measured at each picture point. A non-confocal system that records the decay times in all picture elements simultaneously has been implemented by Lakowicz and Berndt (1991). In a system reported by Kurtz (1987) for studying objects in a continually flowing solution, a single fluorophore is excited using two modulated excitation wavelengths. Two lock-in amplifiers are used in the system, which performs measurements at a single point and produces no images.
Another method to distinguish between fluorophores with different decay times is to use a repetitive source of short optical pulses and employ time-correlated single-photon counting. This technique has also been combined with imaging. A non-confocal system of this kind has been implemented by Morgan and Murray (1991). A confocal system using time gating has been implemented by Buurman et al. (1992).
The term fluorescence lifetime imaging (FLIM) is used for techniques that represent a combination of decay time measurements and imaging, see Lakowicz and Berndt, (1991).
It is an object to provide an improved microfluorometer capable of simultaneously quantifying multiple fluorophores with greater efficiency.
It is another object of the present invention to provide an improved microfluorometer which simultaneously utilizes the entire emissions of multiple fluorophores or the entire emission spectra except possibly for minor parts.
It is a further object to provide an improved microfluorometer capable of separating the individual contributions from fluorophores having overlapping absorption spectra from the combined emission spectrum detected.